As a substrate progresses from the ES complex to the transition state, binding grows stronger by a factor that matches or surpasses the rate enhancement. The principal investigator recently began to explore the rate enhancements that enzymes produce, by measuring the rates of the corresponding reactions in the absence of a catalyst. The results offer a means of recognizing those enzymes that produce the largest rate enhancements, a basis for analyzing the effects of enzyme modification, and a guide to those enzymes that should serve as the most sensitive targets for inhibitor design, useful in designing new transition state analogues as drugs. Dr. Wolfenden proposes to extend those measurements to include each of the major classes of reactions catalyzed by enzymes. In addition to very strong binding of the altered substrate in the transition state, a major challenge faced by an enzyme's active site is to avoid tight binding of the substrate in the ground state. To understand this aspect of enzyme action, he is also investigating the structures of enzyme-product complexes, for comparison with those of enzyme complexes with S* analogues. Finally, he seeks to determine the nature of the detailed forces that are involved in S* binding, which constitute an enzyme's mechanism of action. To determine the nature of those forces, he will continue the structural analysis of ES* complexes, focusing on native and mutant E. coli cytidine deaminase and yeast OMP decarboxylase, both expressed in E. coli. The contributions of active site binding determinants to transition state affinity will be evaluated by examining at different temperatures, the kinetic consequences of truncating the enzyme, substrate or transition state analogue, to evaluate individual group contributions to the free energy, enthalpy and entropy of enzyme-substrate association in the ground state and transition state. In addition, comparison of the transition state binding properties of the native enzyme with those its pieces, obtained by mutation, is expected to reveal the benefit to catalysis that an enzyme gains from being properly connected.